Gene and Protein Expression Profile of Naive and Osteo-Chondrogenically Differentiated Rat Bone Marrow-Derived Mesenchymal Progenitor Cells

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Gene and protein expression profile of naive and osteo-chondrogenically differentiated rat bone marrow-derived mesenchymal progenitor cells

SUSANNE GRÄSSEL1, NAZISH AHMED2, CLAUDIA GÖTTL1 and JOACHIM GRIFKA3

1Experimental Orthopedics, Centre for Biotechnology, BioPark I, University of Regensburg, 93053 Regensburg, Germany; 2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; 3 Department of Orthopedics, University of Regensburg, Kaiser Karl V Allee 3, 93077 Bad Abbach, Germany

Received December 5, 2008; Accepted February 12, 2009

DOI: 10.3892/ijmm_00000188

Abstract. Adult mesenchymal progenitor cells (MPCs) are lysate of MPCs according to their differentiation commitment. adherent stromal cells of non-haematopoietic origin derived Identification of differentially expressed genes and proteins from bone marrow and other tissues. Upon limited in vitro along the osteo-chondrogenic lineage provides a foundation expansion, they retain their self-renewal capacity as well as for a more reproducible and reliable quality and differentiation their potential to differentiate into tissues of mesenchymal control of rat bone marrow-derived MPCs used for osteo- lineage, such as bone, cartilage, muscle, tendon and connective chondrogenic differentiation studies. tissues. Amongst these tissues, cartilage is the only one with insufficient self-renewal capacity, thus MPCs would qualify as Introduction an excellent tool for therapeutic regeneration of focal cartilage lesions. However, optimal in vitro manipulation of MPCs is Adult mesenchymal progenitor cells (MPCs) also known as a prerequisite; identification and a better understanding of the mesenchymal stem cells (MSCs) are precursors of the tissues molecular mechanisms regulating their differentiation of mesenchymal lineage. These osteo-chondroprogenitor pathways are needed. Despite wide usage of rats as a MPCs reside as part of the stroma in the medullary cavity of mammalian experimental model for preclinical fracture the bone (1) and make up ~0.1% of the bone marrow cellular healing and orthopaedic tissue regeneration studies, basal population which is otherwise dominated by haematopoietic gene and protein expression profiles of the osteo-chondrogenic cells. In vitro expansion of bone marrow cells leads to differentiation lineages of adult rat MPCs have rarely been separation of the stromal MPCs based on their capacity of investigated. Therefore, this study was carried out for a adherence to the plastic surface of tissue culture vessels. Upon quantitative RT-PCR based time-course profiling of osteo- and limited expansion these cells retain not only their self-renewal chondrogenesis related gene expression in undifferentiated and capacity, but also their differentiation competence (2). This differentiated rat adult MPCs. In addition, with an antibody intrinsic competence is invoked under controlled nutritional array analysis TIMP-1, MCP-1 and VEGF·-164 were detected and mechanical conditions to differentiate MPCs into bone, in the culture supernatant and CINC-2 and ß-NGF in the cell cartilage, muscle, tendon and connective tissue cells (3). MPCs have already shown promise for ex vivo regenerative medicine ______such as treatment of large bone defects (1,4,5-7) and focal cartilage lesions (8-10). Lack of precise molecular definition and common standards Correspondence to: Dr Susanne Grässel, Department of Orthopedic for initial cell preparations are major obstacles for MPC-based Surgery, Experimental Orthopedics, ZMB/BioPark 1, Josef- research and application. Nevertheless, genetic profiling of Engertstrasse 9, 93053 Regensburg, Germany human MPCs for trans-differentiation capacities has shown E-mail: [email protected] differential gene expression according to origin and commitment status of MPCs (11). In contrast, very little is Abbreviations: MPCs, mesenchymal progenitor cells; TIMP, tissue inhibitor of matrix metalloproteases; MMP, matrix metalloproteases; known about the genomic and proteomic profile of MPCs from VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; other species which may qualify as osteo-chondroprogenitor MCP, monocyte chemoattractant protein; CINC, cytokine-induced cells. Mammalian animal models, like rat, share many aspects neutrophil chemoattractant; NGF, nerve growth factor; Ihh, Indian of the human genomic, cellular and immunological structures. hedgehog; TGF, transforming growth factor; COMP, cartilage Several inbred strains are available; parallel to the small size, oligomeric matrix protein; CNTF, ciliary neurotrophic factor; IFN, rapid development of the animals and high experimental interferon; IL, interleukin; MIP, macrophage inflammatory protein reproducibility are advantages of employing rat MPCs for in vitro studies. Rats are routinely used for fracture healing Key words: mesenchymal progenitor cells, rat, osteogenic, studies, which is a specialized post-natal repair process that chondrogenic, profiling recapitulates embryological skeletal development (12). Besides 745-755 23/4/2009 01:08 ÌÌ Page 746

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studies using genetically engineered MPCs to analyse their rat CD45 (CBL 1502, Chemicon, Germany), rat CD49a (SM effects on healing critical sized fractures (13,14), many reports 805P, Acris, Germany), rat CD71 (#554890) and rat CD106 exist which analyse factors affecting fracture healing and (#559165) (both BD Bioscience Pharmingen, USA). The osteogenic differentiation of precursor cells residing in the CD45-negative, unlabelled fraction was used for further callus during endochondral ossification in the rat model differentiation studies while the CD45-positive, labelled (15-18). Importantly, rats are additionally employed for fraction was discarded. osteogenic and chondrogenic differentiation studies of bone marrow derived or synovial MPCs (19-21). In order to elucidate Chondrogenic and osteogenic differentiation of MPCs. For molecular signalling pathways during fracture healing, a tran- chondrogenesis, cells were cultured for up to 21 days in high scriptional profiling of temporal gene expression rates during density 3-D alginate bead cultures. To prepare the culture callus maturation was performed in the rat model. Here, RNA 107 cells/ml were suspended in 1.2% alginate. The cell-alginate

was isolated from total callus tissue which contained a mixture amalgam was dropped into 102 mM CaCl2 solution via a of not well defined cells in various differentiation stages (22). syringe which resulted in a formation of beads with a However, genomic and proteomic profiling of rat CD45- diameter of 2-3 mm containing ~105 cells/bead. Beads were depleted MPCs during progression along the osteogenic/ cultured in 2.3 ml chondrogenic medium in 12-well tissue chondrogenic differentiation pathway is either incomplete or culture plates. Ten alginate beads per culture set up were unavailable. used for RNA isolation and gene expression analysis. Cells Therefore, this study undertook mRNA level profiling of were released from alginate by incubation at 37˚C for 30 min selected genes in undifferentiated adult rat MPCs defined as in 55 mM sodium citrate and 0.15 M sodium chloride buffer baseline gene expression profile. Changes in gene regulation followed by cell recovery with a 3 min spin at 750 x g. were then evaluated in an in vitro time-course study during Chondrogenic medium contained, ITS+ premix 6.25 μg/ml osteo-chondrogenic differentiation. insulin, 6.25 ng/ml selenium acid, 6.25 μg/ml transferrin, Additionally, a protein profile of selected growth factors 1.25 mg/ml BSA and 5.35 μg/ml linoleic acid (BD and cytokines generated from undifferentiated MPCs was Biosciences), 110 μg/ml pyruvate, 40 μg/ml proline, 0.1 μM compared with that of cells which were differentiated along dexamethasone, 50 μg/ml ascorbic acid and 10 ng/ml TGFß-3 osteogenic and chondrogenic lineages. (24) (R&D Systems) in ·-MEM high glucose medium (Gibco, Establishing a differential, temporal gene and protein Invitrogen). Alginate sodium salts were acquired from Sigma expression profile allowed identification of markers suitable Aldrich. For osteogenesis, 2.5x105 cells/well were cultured for for monitoring progression of rat MPCs along the osteogenic up to 21 days in 6-well culture plates as monolayer in osteo- and chondrogenic pathway. This knowledge is essential for genic medium containing 10% FCS, 1% pen/strep, 10 nM providing proper and optimized conditions for in vitro dexamethasone, 50 μg/ml ascorbate 2-PO4 and 10 mM ß-Na- differentiation studies of MPCs. glycerophosphate (Sigma Aldrich) in ·-MEM high glucose medium. Materials and methods Immunofluorescence. For immunofluorescence analysis, Preparation of MPCs and cell culture. MPCs were isolated alginate beads were irreversibly polymerized by replacing

from rat bone marrow as described earlier (23). Briefly, bone CaCl2 with 100 mM BaCl2 (25). The beads were fixed with marrow was removed from the tibiae and femora of 6-week old 4% paraformaldehyde and after sequential dehydration male Sprague-Dawley rats by centrifugation (800 x g for 3 min embedded in paraffin. Sections (4 μm) were acquired and the in an Eppendorf mini-centrifuge). Homogenized bone marrow deparaffinized and rehydrated sections were used for staining was cultured in 175 cm2 tissue culture flasks in proliferation after hyaluronidase digestion for 6 min at RT. Undifferentiated medium containing 5% glutamate, 1% antibiotics/antimycotics MPCs kept in monolayer culture were fixed with 4% para- and 10% FBS (Gibco, Invitrogen, UK) in ·-MEM (Sigma formaldehyde. Slides were blocked for 1 h at 37˚C in 5% Aldrich, Germany). Non-adherent cells were removed on the normal goat serum and 1% BSA in PBS containing Complete 3rd day and the adherent CFU-cells were proliferated until Mini 1:5 protease inhibitor solution (Roche, Germany). After 70% confluency. Magnetic associated cell sorting procedure washing with PBS, cells were stained overnight at 4˚C with (MACS, Miltenyi Biotech, Germany) was carried out according monoclonal antibodies which recognize rat collagen II, diluted to the manufacturer's instructions. In short, expanded cells from 1:100, and rat D7fib, diluted 1:50 (both from Acris) and 3-5 different rats were incubated in suspension with 4 μl of CD49a, diluted 1:50 (Chemicon). The appropriate Alexa 568 selected antibody/106 cells for 5 min at 37˚C followed by or 488 conjugated secondary antibodies (goat anti-mouse, washing and incubation with goat anti-mouse secondary 5 μg/ml, Molecular Probes, USA) were added for 1 h at RT. antibody coupled with magnetic beads for 15 min at 4˚C. After washing, slides were permanently mounted with This suspension was passed through a magnetic column fluorescent mounting medium (Dako, USA) and covered (Miltenyi Biotec's LS-MACS columns). While labelled cells with cover slips. Slides were evaluated with a scanning laser were retained by the magnetic field, the flow through containing microscope (C1 confocal microscope from Nikon, Germany) the unlabelled negative fraction was collected by washing the and photos were taken with a Nikon C4 camera and software. columns with buffer. After removing the column from the magnetic field, the antibody labelled cells (positive fraction) Histological analysis. Osteogenically differentiated cells were were also flushed out. For both fractions cells were counted washed with PBS and fixed with methanol for 10 min. After and stained. Antibodies used for MACS were directed against rinsing with water, staining was carried out for 2 min with 1% 745-755 23/4/2009 01:08 ÌÌ Page 747

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Table I. Primers used for quantitative RT-PCR. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Gene NIH Amplicon Forward primer Reverse primer accession no. (bp) (5'-3') (5'-3') ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– ·10 XM_001063132 188 -tttcttcgggaaatcagagc -tggatggagaagccaatctc ·11 XM_001075650 170 -tggaggtccaacacttcctc -gggtttcagtccctcctctc Aggrecan NM_022190 224 -ggccttccctctggatttag -ccgcactactgtccaac ß-Actin NM_031144 104 -gtagccatccaggctgtgtt -ccctcatagatgggcagagt CINC-2 D87926 287 -cactgcttctgctgcttctg -tgacttctgtctgggtgcag Col1a1 RGD61817* 59 -tccagggctccaacgaga -ctgtaggtgaatccactgttgc Col2a1 NM_012929 60 -cccctgcagtacatgcgg -ctcgacgtcatgctgtctcaag Col10a1 AJ131848* 247 -ccctattggaccaccaggta -tctctgtccgctctttgtga Col16a1 M92642 97 -gcctggtaccaaaggtgaaa -catagcctggaggaccttga COMP NM_012834 167 -tgacttcgatgctgacaagg -gaacgatctccattccctga Ihh XM_343590 103 -atgaagacggccatcactcag -cgcgccagtagtccgtacttat MCP-1 M57441 167 -atgcagttaatgccccactc -ttccttattggggtcagcac MMP-2 NM_031054 111 -gaccggtttatttggcgga -ggcctcatacacagcgtcaat MMP-13 XM_343345 93 -acctgggatttccaaaagagg -acacgtccttccctgagaaga ß-NGF XM_001067130 182 -ggacgcagctttctatcctg -ctgtgtcaagggaatgctga Runx2 XM_34016 86 -gccgggaatgatgagaacta -agatcgttcaacctggccact Sox4 XM_344594 58 -ggcccatgaacgcctttat -ctggatgaacgggatcttgtc Sox6 XM_215016 51 -gaaatccatgtccaaccaggac -cgggcctgctcttcatagtaag Sox9 XM_343981 140 -ctgaagggctacgactggac -tactggtctgccagcttcct Tbox2 XM_220810 71 -gcccactctccgtttgtatgag -aggacgaggcatcggattc TIMP-1 NM_053819 136 -gattcgacgctgtgggaaat -tttccgttccttaaacggcc TIMP-2 NM_021989 140 -ggcaagatgcacattaccctct -atgtagcatgggatcatagggc TGFß-3 NM_013174 86 -ttccttcttggccgtatttcc -tgtgtgggatccagaatcca VEGF· NM_031836 71 -tggctttactgctgtacctcca -tttctgctccccttctgtcgt VEGFR-2 NM_013062 95 -ttgcctagtcaagcagctcgt -cgatggtctcaccaatggttg ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– *Locus ID. –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

alizarin red prepared in 25% ammonia. Stained cells were Systems). Amplification parameters were identical for all thoroughly washed and photographed with a Nikon C4 camera primer pairs and were repeated for 40 cycles, denaturation after complete drying. occurred at 95˚C for 0.15 min and annealing at 60˚C for 1 min. Mean relative quantification (RQ) values were calculated by RNA isolation and reverse transcription. RNA was isolated by the software ‘RQ study application v1.1’ (ABI Prism 7000 SDS an affinity column chromatography method with Ambion's software v1.1) according to the ΔΔCt method using ß-actin RNAqueous4-PCR kit according to the manufacturer's as endogenous control and undifferentiated MPCs (day 0) as protocol. For removal of putative DNA contamination calibrator. Primers were designed either with freeware Primer3 DNaseI enzyme (DNA-free, Ambion, USA) was used. RNA (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) concentration was determined at a 585 nm wavelength with or ‘Primer Express’ software supplied by AB Systems. All RiboGreen RNA quantification kit (Molecular Probes). RNA primers were manufactured at MWG-Biotech, Germany and (0.5-1 μg) was converted to cDNA with SuperScript II are listed in Table I. Prior to gene expression analysis, primer reverse transcriptase kit (Invitrogen) in 20 μl of total reaction efficiency was determined with 10(1-S)-1, where S is the slope volume in the presence of 40 units/μl recombinant ribonuclease of the curve of a cycle threshold (Ct) standard curve. Only inhibitor (RNase OUT®), 500 μg/ml of Oligo-dT primers, 90-100% efficient primers were used for analysis (26) and 10 mM dNTPs and 200 units of SuperScriptII enzyme in efficiency was included into calculation of gene expression First-Strand Buffer and 0.1 M DTT for 50 min at 42˚C changes according to the method of Pfaffl et al (27). For each followed by an extension period of 15 min at 70˚C. primer pair cDNA (not pooled) from 4-7 rats was used for PCR. Relative quantitative PCR. Relative quantitative PCR was performed using the SYBR Green Dye I on ABI 7000 Prism Antibody array analysis. RayBio™ (Tebu-bio, France) rat Sequence detection system (AB Systems, USA) according to cytokine antibody array I was used according to the the manufacturer's instructions. Briefly, 1 μl of cDNA was manufacturer's instructions. Briefly, membranes were blocked amplified in triplicates in 50 μl final volume containing 0.2 μM for 30 min in 5% BSA in 0.01 M Tris buffer with 0.15 M of each primer suspended in SYBR Green master mix (AB NaCl (pH 7.6) followed by overnight incubation at 4˚C in 1 ml 745-755 23/4/2009 01:08 ÌÌ Page 748

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Figure 2. Antigen profiling of MPCs. Separation of bone marrow-derived adherent cells by magnetic antibody labelling resulted in a mixture of 60- 75% CD71-, CD49a- and CD106-positive cells. About 85% of the cells were CD45-negative stromal cells indicating a slightly heterogeneous population with ~15% of CD45-positive haematopoietic cells. n=3-5 independent experiments.

Figure 1. Morphological characterisation of MPCs. CFU-F like marrow derived adherent cells (a) characterized by double-immunofluorescence staining with D7fib (red fluorescence) and CD49a (green fluorescence) (b) like morphology (Fig. 1a). Immunofluorescence analysis of exhibited typical stem cell morphology. At day 21 after induction of the adherent cells exhibited positive staining for fibroblast osteogenic differentiation in monolayer the cells were stained with alizarin marker D7fib which is described as a specific marker for red. Positive staining indicates calcified matrix formation (d) while the MPCs as suitable as STRO-1 and CD105 (30). Positive control culture kept in proliferation medium remained unstained (c). Chondrogenically differentiated cells kept in alginate beads were stained staining was also detected for ·1 integrin marker CD49a with an antibody against collagen II. After 21 days in 3D-culture the (Fig. 1b). To confirm that isolated cells are indeed MPCs, extracellular matrix displayed a positive collagen II signal in the cell cells were tested for their retained differentiation potential surrounding newly synthesized matrix (f) which was undetectable at day 0 after expansion in vitro. MPCs were induced for up to 21 days of 3D-culture (e). in monolayer culture to undergo osteogenesis and, in 3-D high density alginate cultures for chondrogenesis. Osteogenic cultures stained positive for alizarin red indicating formation of of cell culture supernatant or cell lysates containing 100 μg a calcified matrix while uninduced control cultures remained of total protein. Both, cell culture supernatant and cell lysates unstained (Fig. 1c and d). Chondrogenically induced cells were harvested at the endpoint (21 days) of culture duration. exhibited extensive collagen II staining in the surrounding Cell lysates were prepared by using cell lysis buffer provided matrix on day 21 of the culture which was undetectable at by the manufacturer. After washing and incubation with biotin day 0 (Fig. 1e and f). coupled anti-cytokine antibodies for 2 h a second series of Since a unique MPC marker is not yet identified, culture washing was performed and then the membranes were purity is usually determined by employing a consortium of incubated with horseradish peroxidase-conjugated streptavidin positive and negative markers. In this study adherent cells were for 2 h. Signals were detected with the provided detection screened by magnetic cells sorting (MACS™) using the three solution using a CURIX 60 film developer (Agfa, Germany). MPC-positive markers CD71 (transferrin receptor) (31), CD106 (VCAM) (32) and CD49a (integrin subunit ·1) (33,34) (Fig. 2). Statistics. Changes in gene expression <2-fold were not These markers reveal 60-65% of the cells as CD71 positive considered physiologically relevant and therefore described and 75% positive for CD106 and CD49a. In addition, CD45 as unchanged (27,28) even if statistically significant. (leukocyte common antigen) (35,36), a haematopoietic surface Expression rates of genes were compared between the two marker used for negative selection, revealed strong enrichment groups (undifferentiated and differentiated) using one- of MPCs with a remaining 15% of CD45-positive cells. repeated measure ANOVA with the assumption of homo- geneity of variance used as appropriate. Only where a statistical Gene expression profiling of undifferentiated and differentiated significance was found, pair-wise comparisons of the groups MPCs. With the help of quantitative RT-PCR and specific were performed using the t-test. A p-value <0.05 was primers (Table I) mRNA expression of selected genes was considered statistically significant. determined in un-induced, undifferentiated MPCs (day 0) and defined as baseline or calibrator gene expression (data not Results shown). After application of osteogenic or chondrogenic favourable Cellular morphology and surface antigen marker profile of conditions to the cell cultures for up to 21 days, relative MPCs. Formation of CFU-F is one of the basic classifications changes in mRNA level were determined on days 7, 14 and 21 of bone marrow derived stem cells along with rapid adhesion (Fig. 3A-H). During induction of chondrogenic differentiation, and extended proliferation capacity (29). After in vitro tissue inhibitor of matrix metalloproteases (TIMP-1) became expansion of bone marrow isolated cells, colony forming units downregulated at day 21 while TIMP-2, MMP-2 and -13 of fibroblast like cells (CFU-F) designated as MPCs readily remained upregulated until day 21 (Fig. 3A). Integrin subunit adhered to the culture flasks and showed typical fibroblast- ·11 was upregulated non-physiologically (2x) according to 745-755 23/4/2009 01:08 ÌÌ Page 749

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Figure 3. Differential gene expression profile of MPCs during osteo-chondrogenic differentiation. Relative quantitative mRNA expression level (RQ) of selected TIMPs, MMPs, Integrin subunits, collagens, transcription factors and growth factors was determined by the ΔΔCT method using ß-actin gene expression as endogenous control and gene expression of undifferentiated MPCs as calibrator which was set at 1. Expression level of each gene was determined at day 7 (white bars), day 14 (grey bars) and day 21 (black bars) during chondrogenic (A, C, E, G) and osteogenic (B, D, F, H) differentiation of MPCs. n=4-7 independent experiments. 745-755 23/4/2009 01:08 ÌÌ Page 750

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Table II. Regulation of gene expression during osteo-chondrogenic differentiation of MPCs at day 21 (end point of culture regimen) expressed as relative quantification values (RQ). ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Gene RQ chondrogenesis RQ osteogenesis Ratio RQ of chondrogenesis/osteogenesis ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Col2a1 30818.00*** 0.20*** 181276.00 Col10a1 808.00*** 2.00 n.s. 404.00 Ihh 103.00*** 0.40*** 274.00 Aggrecan 32.00*** 0.30*** 107.00 VEGFR-2 2.70** 0.04*** 67.50 Integrin ·10 85.00*** 1.50*** 57.00 Sox6 20.50*** 0.40*** 51.00 Col1a1 50.00*** 1.00 n.s. 50.00 COMP 897.00*** 28.00*** 32.00 Sox9 26.00*** 1.00 n.s. 26.00 CINC-2 1.50*** 0.15*** 10.00 MMP-13 30.00*** 3.00*** 10.00 Runx2 6.00*** 0.70* 8.60 VEGF · 2.40*** 0.35*** 6.90 Tbox2 6.70*** 1.35*** 5.00 Col16a1 2.80*** 0.70*** 4.00 Sox4 6.50*** 3.25*** 2.00 MMP-2 3.60*** 2.50*** 1.40 Integrin ·11 2.00*** 1.60*** 1.25 TIMP-2 6.20*** 23.60*** 0.30 TIMP-1 0.40*** 1.40* 0.30 ß-NGF 0.20*** 0.60*** 0.30 TGFß-3 0.40*** 2.80*** 0.14 MCP-1 0.05*** 1.00 n.s. 0.05 ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Values >1 indicate upregulation, values <1 indicate downregulation of the gene relative to the calibrator (undifferentiated MPCs set as 1). *p<0.05; **p<0.01; ***p<0.001; n.s., non-significant. –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Pfaffl (27,28) while integrin subunit ·10 became strongly and ·11 remained below physiological relevance. Contrary to induced. Gene expression of TGFß-3 was downregulated chondrogenesis TGFß-3 mRNA level was moderately up- from day 7 while vascular endothelial growth factor (VEGF) · regulated during osteogenic culture time (2.8x) while both and VEGFR-2 mRNA level were moderately increased at the VEGF· and VEGFR-2 mRNAs were downregulated, gene end of culture time after an earlier suppression (Fig. 3C). Gene expression of VEGFR-2 decreased 25x compared to its expression of transcription factors Sox9, Sox6, Sox4, Tbox2, expression in undifferentiated MPCs (Fig. 3D). Gene expression Runx2 and Ihh was clearly upregulated with an increase profiles of all transcription factors (except for Sox4) was towards the culture end point (Fig. 3E). Notably, Ihh mRNA different under osteogenic compared to chondrogenic culture level increased up to 100x at day 21. All collagens were conditions. Sox9 was downregulated at day 7 while at the end upregulated during culture time with a profound increase in of culture time gene expression was similar to undifferentiated gene expression for Col2a1 (>30,000x) and the lowest change MPCs. Sox6 and Ihh mRNA level were strongly suppressed for Col16a1 (2.8x). Gene expression of aggrecan and COMP towards later time points while Tbox2 remained unaltered and was strongly induced over culture time (Fig. 3G). Runx2 was slightly downregulated (Fig. 3F). Also, collagen Induction of osteogenic differentiation resulted in a mostly gene expression profiles were different from chondrogenic altered expression profile of these genes. TIMP-1 was not induced MPCs. During osteogenesis Col1a1 mRNA level did regulated in physiologically relevant terms (<2x) while gene not change compared to undifferentiated MPCs while Col2a1 expression of TIMP-2 was more profoundly upregulated at day was profoundly downregulated during progression of osteo- 21 than in chondrogenic differentiation (23x versus 6x). genesis. Col10a1 was upregulated until day 14 and almost MMP-2 gene expression was similarly regulated as under reduced to the level of uninduced MPCs at day 21. Gene chondrogenic differentiation while MMP-13 became down- expression of Col61a1 and aggrecan decreased over culture regulated at the end of culture compared to chondrogenic time while COMP became upregulated, however, less conditions (3x versus 30x) (Fig. 3B). During osteogenic profoundly as under chondrogenic differentiation conditions differentiation changes in expression for integrin subunits ·10 (28x versus 900x) (Fig. 3H). Regulation data from the end 745-755 23/4/2009 01:08 ÌÌ Page 751

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Figure 4. Comparison of cytokine and growth factor profiles of undifferentiated and differentiated MPCs. An antibody array containing 14 different cytokines plus TIMP-1, ß-NGF, VEGF·-165, leptin and CNTF was used to compare protein profiles of proliferated undifferentiated, uninduced MPCs and MPCs after osteogenic and chondrogenic induction (A). *TIMP-1, **VEGF·-164, ΔMCP-1, ∞ß-NGF, +CINC-2. Representative blots of a total of 5 independent experiments are shown; n=3 for culture supernatants and n=2 for cell lysates. Temporal gene expression profiles of MCP-1, CINC-2 and ß-NGF was determined by qRT-PCR as described above (B and C).

point of the different culture regimen (day 21) are displayed (CNTF), fractalkine, GM-CSF, IFN-Á, IL-1·, -1ß, -4, -6, -10, in Table II. lipopolysaccharide induced c-x-c chemokine (LIX), leptin, macrophage inflammatory protein (MIP-3·) and TNF-·. Cytokine and growth factor protein profile of undifferentiated Gene expression level of MCP-1 was profoundly down- and differentiated MPCs. Cell culture supernatants and lysates regulated during chondrogenic differentiation while from undifferentiated and differentiated MPCs at day 21 were unchanged along osteogenic differentiation pathway expression. analyzed for secretory molecules using an antibody array CINC-2 was downregulated upon osteogenic differentiation covering 14 cytokines plus ß-NGF, TIMP-1, CNTF, leptin and and remained unaltered during chondrogenic differentiation VEGF·-164 (Fig. 4). TIMP-1 was the only molecule secreted (<2x at day 21) while gene expression of ß-NGF was decreased from undifferentiated as well as differentiated MPCs, while upon induction of MPC differentiation (Fig. 4B and C). the cytokine monocyte chemoattractant protein (MCP-1) was exclusively secreted from undifferentiated MPCs. Secretion RQ values and ratios. Table II summarizes regulation of of VEGF (variant VEGF·-164) was observed only after gene expression at the end point of the osteo-chondrogenic induction of osteogenic and chondrogenic differentiation. differentiation culture regimens (day 21). Opposite regulated When the same antibody array was examined with the cell genes include Col2a1, Ihh, aggrecan, VEGFR-2, Sox6, Runx 2, lysate two additional molecules were identified. Nerve VEGF·, Col16a1 and TGFß-3. Except for TGFß-3, the other growth factor (ß-NGF) was detected in undifferentiated and genes listed above were downregulated upon induction of osteogenically induced cells only and cytokine induced osteogenesis and upregulated upon chondrogenesis. Genes neutrophil chemoattractant (CINC-2) was detected as a weak that are not regulated upon osteogenesis but upregulated spot after osteogenesis. Both factors were undetectable after upon chondrogenesis were Col10a1, integrin subunit ·10, chondrogenic induction (Fig. 4A). The remaining 14 proteins Col1a1, Sox9, Tbox2, integrin subunit ·11 while TIMP-1 and of the array were undetectable in all culture conditions MCP-1 were downregulated upon chondrogenesis. Only analysed, these include, CINC-3, ciliary neurotrophic factor CINC-2 mRNA level was suppressed in osteogenesis and 745-755 23/4/2009 01:08 ÌÌ Page 752

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remained unchanged upon chondrogenesis. Genes upregulated expression levels become induced in MPCs after entering the under both osteogenic and chondrogenic differentiation chondrogenic pathway while commitment to osteogenic conditions include COMP, MMP-13, Sox4, MMP-2 and differentiation regimen results in strong downregulation of TIMP-2. The only gene downregulated under both differentiation VEGF· and VEGFR-2 mRNA. Hence, comparison of relative pathways was ß-NGF. VEGF· and VEGFR-2 mRNA levels are useful indicators of The ratio of RQ reflects whether the gene is up- or down- MPC differentiation stage and lineage commitment. regulated in chondrogenesis versus osteogenesis. Values >1 Transcription factors which regulate osteo- and chondro- indicate upregulation of mRNA levels during chondrogenesis genic lineage commitment as, Tbox2 (brachyury), Sox9, Sox6 while values <1 indicate downregulation during chondrogenesis and Runx2 are clearly upregulated during chondrogenic compared to osteogenesis. differentiation. Both, Sox9 and Tbox2 are essential components for the BMP-dependent onset of chondrogenesis (40). Except Discussion for Sox4, the other four transcription factors were differentially regulated under osteogenic culture conditions. Differential Rat is an appropriate animal model for MPC based pre-clinical expression levels of these transcription factors might serve as studies due to high reproducibility and at the same time markers for harvesting osteo-chondroprogenitor cells at provides a sufficient number of bone marrow derived cells. different stages of lineage commitment thus allowing However, a representative gene expression profile of rat naïve reproducible separation and identification of these early and differentiated MPCs is not available and as the expression committed cells. Similarly, Sox4 was not implicated in being of known genes is not identical to adult human stem cells, a involved in the osteogenic differentiation pathway and not comprehensive profiling of rat gene and protein expression is much is known regarding its involvement in chondrogenic necessary for accurate interpretation of data generated with differentiation except that Sox4 and BMP-2, together with rat MPCs. collagens I and IV are upregulated in early chondrogenic Here, we present a time course profiling of a selected panel differentiating human MPCs kept in alginate beads (41). of genes mostly differentially regulated in undifferentiated and During chondrogenic differentiation, gene expression of both osteo-chondrogenically differentiated rat MPCs. These genes Sox9 and Ihh is strongly induced, indicating cellular arrest in include growth factors, transcription factors, metalloproteinases the early hypertrophic stage. Sox9 is expressed in all essential (MMPs) and their inhibitors (TIMPs), structural components of stages of chondrogenic development, but its expression is the matrix and cell adhesion molecules. Several of these genes abrogated in hypertrophic chondrocytes (42,43) which may routinely serve as markers to determine osteo-chondrogenic be regulated by a negative feedback mechanism of the PTHrP differentiation status of human chondrocytes, osteocytes and and Ihh signal cascade in pre-hypertrophic chondrocytes MPCs (col1a1, col2a1, col10a1, Runx2, Sox9, aggrecan) others (44,45). However, this expression profile is accompanied by are not yet implicated in osteo-chondrogenic differentiation of strong upregulation of collagen X gene expression which is MPCs (Col16a1, Sox4, TIMP1, -2, MCP-1, ß-NGF and restricted to terminally differentiated hypertrophic chondro- CINC-2). Not much is known about the temporal involvement cytes (46). Possibly, upregulation of collagen X is connected of all of these markers in osteo-chondrogenic differentiation to increased expression of Runx2 since multiple Runx2 processes of rat MPCs and whether their regulation is consensus binding elements are located in the collagen X comparable to the human system. promoter region (47). However, collagen X expression in vitro is not in line with the expression of other differentiation Identification of genomic marker for monitoring osteogenesis markers. This limitation in using collagen X as a marker for and chondrogenesis. Integrin subunit ·11, expressed in chondrogenesis of human MPCs is described in previous undifferentiated rat MPCs, is present in various mesenchymal literature (48,49). Our data suggest the same for rat MPCs tissues around the cartilage anlage in the developing skeleton. during chondrogenic differentiation. In comparison, integrin subunit ·10, known as a highly MMPs alter the activity or function of numerous proteins chondrocyte specific, collagen II and IX-binding integrin by proteolytic processing and they are also involved in various (37,38), was not detected in naïve MPCs. Chondrogenic cellular functions and matrix formation during development. differentiation of MPCs leads to ~85-fold increase in the Expression of MMPs and TIMPs in MPC cultures was mRNA level of ·10, whereas the ·11 mRNA level remains reported on in earlier studies on human MPCs of different mostly unchanged. Gene expression profile of both integrin origins (50,51). MMP-13 together with its inhibitors, TIMP-1 subunits remained unaltered during the osteogenic and -2 is expressed in MPCs indicating a role in stem cell differentiation pathway. Thus, the ratio of ·11/·10 gene migration and proliferation as it is assigned to MMP-2 (52). expression is highly appropriate for monitoring the progression While TIMP-1 gene expression remains mainly unaltered or of chondrogenic versus osteogenic differentiation of MPCs. is downregulated, MMP-13 and TIMP-2 were upregulated Gene expression profiles of growth factors TGFß-3 and during both, chondrogenic and osteogenic culture regimen. A VEGF·, including its receptor VEGFR-2, are differentially relatively higher MMP-13 mRNA level during chondro- regulated upon induction of osteo-chondrogenically genesis might obtain relevance in progressing mineralization differentiation. as MMP-13 is involved in endochondral and intramembranous During endochondral bone formation angiogenic factor ossification (53). VEGF modulates matrix mineralization in hypertrophic We report, for the first time, on gene expression of cartilage in an autocrine manner by upregulating the expression collagen XVI in undifferentiated MPCs which is upregulated of its receptor (39). Here, VEGF· and VEGFR-2 gene during chondrogenesis while downregulated during osteo- 745-755 23/4/2009 01:08 ÌÌ Page 753

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genesis. This observation indicates an unknown role of this genic differentiation but keep it under osteogenic favourable fibril-associated collagen with interrupted triple helices conditions which suggests a role of ß-NGF in osteogenesis as (FACIT) in proliferation of un-induced MPCs and/or a trophic factor known to stimulate collagen synthesis and chondrogenic differentiation. As known for human MPCs, expression of specific osteogenic marker, such as alkaline collagen II and aggrecan gene expression are upregulated phosphatase (63). during chondrogenesis and reduced in osteogenesis, therefore The discrepancy in reduced gene expression and protein their differential expression profiles obtained in this study are synthesis/secretion profile of VEGF·, ß-NGF and CINC-2 after also usable for rat MPCs as marker of chondrogenic differentiation is likely due to post-translational regulation. commitment. VEGF production is known to be regulated at the translational level as over-expression of transcription factor c-Myc related Identification of proteomic marker for monitoring osteogenesis with rapid induction of tumors, also leads to 10-fold higher and chondrogenesis. Secretory cytokines, proteases and growth VEGF production while the gene expression level remains factors are key players in cell proliferation and differentiation. unaltered. The mechanisms of action are most likely higher Therefore, we investigated MPC differentiation state-specific ribosome biogenesis rates which increase translation efficiency protein profile. TIMP-1, the only identified molecule secreted of selected mRNAs (64). Also, in rat smooth muscle cells from undifferentiated as well as differentiated MPCs is a multi- regulation of NGF production does not solely occur at the functional molecule. In addition to its physiological activity level of transcription. Post-transcriptional mechanisms, such as as inhibitor of MMPs, it is also thought to be involved in increased NGF mRNA stability and elevated synthesis of NGF regulation of cell growth and differentiation of a variety of cell protein per unit NGF mRNA, can operate (65,66). types including mesenchymal and epithelial cells (54). Hence, In conclusion, we demonstrated a specific gene and protein TIMP-1 secreted differentiation independently of MPCs act expression/secretion profile of rat CD45-low bone marrow- either as a growth factor or as an inducer/repressor of specific derived progenitor cells. The defined single genes and gene factors necessary for commitment to the osteo-chondrogenic combinations are considered suitable as markers allowing lineage of MPCs. reliable and reproducible discrimination between the un- MCP-1, which recruits monocytes into ischemic tissue induced MPCs and the osteogenically and chondrogenically and stimulates chemotaxis, is secreted exclusively from un- differentiating MPCs (Table II). Discrimination is carried out induced MPCs while its gene expression remains unchanged also at protein level with the help of the two cytokines MCP-1 or is repressed during osteo-chondrogenic differentiation, and CINC-2 indicative for un-induced and osteogenically respectively. It is speculated to be involved in homing of differentiated MPCs, respectively. The knowledge of circulating stem and progenitor cells. MPCs secrete this differentially expressed genes and proteins provides a chemokine to the region of injuries which contribute to foundation for optimizing cell culture conditions necessary to healing processes (55,56). MCP-1 secretion appears suitable properly induce and maintain desired differentiation pathways. as a marker for un-induced rat MPCs not having entered the This is a prerequisite for not only manipulating MPCs in chondrogenic and osteogenic differentiation cascade. animal models to regenerate complex tissues such as cartilage Secretion of the rat-specific splice variant of VEGF, and bone but also for future tissue engineering studies with VEGF·-164, was induced only after cells were subjected to human adult MPCs. osteogenic and chondrogenic differentiation. VEGF as a multi- faceted protein has a role in induction of metalloproteinases, Acknowledgements such as MMP-13 during the late stages of chondrogenesis marking the onset of hypertrophy (57). Lack of VEGF secretion We thank Dr Evy Lundgren-Akerlund (Cartella AG, in undifferentiated MPCs are taken as an indicator for a Sweden) for providing ·10 and ·11 primers. We also relatively pure MPC population, mostly devoid of VEGF thank Maren Marschner for her excellent technical support. secreting haematopoietic and endothelial progenitor cells This work was supported by a DFG grant assigned to SG (58,59). (GR 1301/7-1). Analysis of the cellular lysates leads to the identification References of two additional molecules. ß-NGF, detected in undifferentiated as well as in cells after osteogenic induction and cytokine- 1. Cancedda R, Dozin B, Giannoni P and Quarto R: Tissue induced neutrophil chemoattractant (CINC-2), which was engineering and cell therapy of cartilage and bone. Matrix Biol detected after osteogenesis only. CINC-2 attracts neutrophils 22: 81-91, 2003. 2. Friedenstein AJ, Gorskaja JF and Kulagina NN: Fibroblast and was suggested to inhibit acute inflammation indicating precursors in normal and irradiated mouse hematopoietic an anti-inflammatory role in osteogenesis (60). So far, this organs. Exp Hematol 4: 267-274, 1976. chemokine has not been determined in rat MPCs undergoing 3. Bruder SP, Fink DJ and Caplan AI: Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration osteogenic differentiation, however, our data suggest that therapy. J Cell Biochem 56: 283-294, 1994. CINC-2 is suitable as one of several reliable protein indicators 4. Quarto R, Mastrogiacomo M, Cancedda R, Kutepov SM, for successfully induced osteogenesis in MPCs. Mukhachev V, Lavroukov A, Kon E and Marcacci M: Repair of large bone defects with the use of autologous bone marrow Undifferentiated, non-committed bone marrow derived cells stromal cells. N Engl J Med 344: 385-386, 2001. are known to express ß-NGF and its receptor (61) together 5. Horwitz EM, Prockop DJ, Fitzpatrick LA, Koo WW, Gordon PL, with other neurotrophic factors (62). This falls in line with Neel M, Sussman M, Orchard P, Marx JC, Pyeritz RE and Brenner MK: Transplantability and therapeutic effects of bone the observed production of ß-NGF in undifferentiated MPCs. marrow-derived mesenchymal cells in children with osteogenesis The naïve MPCs loose ß-NGF biosynthesis upon chondro- imperfecta. Nat Med 5: 309-313, 1999. 745-755 23/4/2009 01:08 ÌÌ Page 754

754 GRÄSSEL et al: GENE AND PROTEIN EXPRESSION PROFILE OF MPCs

6. Mao JJ: Stem-cell-driven regeneration of synovial joints. Biol 28. Bustin SA, Benes V, Nolan T and Pfaffl MW: Quantitative real- Cell 97: 289-301, 2005. time RT-PCR-a perspective. J Mol Endocrinol 34: 597-601, 2005. 7. Raghunath J, Salacinski HJ, Sales KM, Butler PE and 29. Bianco P, Riminucci M, Gronthos S and Robey PG: Bone Seifalian AM: Advancing cartilage tissue engineering: the marrow stromal stem cells: nature, biology, and potential application of stem cell technology. Curr Opin Biotechnol 16: applications. Stem Cells 19: 180-192, 2001. 503-509, 2005. 30. Jones EA, Kinsey SE, English A, Jones RA, Straszynski L, 8. Wakitani S, Imoto K, Yamamoto T, Saito M, Murata N and Meredith DM, Markham AF, Jack A, Emery P and McGonagle D: Yoneda M: Human autologous culture expanded bone marrow Isolation and characterization of bone marrow multipotential mesenchymal cell transplantation for repair of cartilage defects in mesenchymal progenitor cells. Arthritis Rheum 46: 3349-3360, osteoarthritic knees. Osteoarthritis Cartilage 10: 199-206, 2002. 2002. 9. Litzke LE, Wagner E, Baumgaertner W, Hetzel U, 31. Jefferies WA, Brandon MR, Williams AF and Hunt SV: Analysis Josimovic-Alasevic O and Libera J: Repair of extensive of lymphopoietic stem cells with a monoclonal antibody to the articular cartilage defects in horses by autologous chondrocyte rat transferrin receptor. Immunology 54: 333-341, 1985. transplantation. Ann Biomed Eng 32: 57-69, 2004. 32. Buravkova LB and Anokhina EB: Effect of hypoxia on stromal 10. Wilke MM, Nydam DV and Nixon AJ: Enhanced early chondro- precursors from rat bone marrow at the early stage of culturing. genesis in articular defects following arthroscopic mesenchymal Bull Exp Biol Med 143: 411-413, 2007. stem cell implantation in an equine model. J Orthop Res 25: 33. Deschaseaux F, Gindraux F, Saadi R, Obert L, Chalmers D and 913-925, 2007. Herve P: Direct selection of human bone marrow mesenchymal 11. Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U, stem cells using an anti-CD49a antibody reveals their low Krause U, Blake J, Schwager C, Eckstein V, Ansorge W and phenotype. Br J Haematol 122: 506-517, 2003. Ho AD: Comparative characteristics of mesenchymal stem cells 34. Gindraux F, Selmani Z, Obert L, Davani S, Tiberghien P, from human bone marrow, adipose tissue, and umbilical cord Herve P and Deschaseaux F: Human and rodent bone marrow blood. Exp Hematol 33: 1402-1416, 2005. mesenchymal stem cells that express primitive stem cell markers 12. Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT and can be directly enriched by using the CD49a molecule. Cell Einhorn TA: Fracture healing as a post-natal developmental Tissue Res 327: 471-483, 2007. process: molecular, spatial, and temporal aspects of its regulation. 35. Woollett GR, Barclay AN, Puklavec M and Williams AF: J Cell Biochem 88: 873-884, 2003. Molecular and antigenic heterogeneity of the rat leukocyte- 13. Tsuchida H, Hashimoto J, Crawford E, Manske P and Lou J: common antigen from thymocytes and T and B lymphocytes. Engineered allogeneic mesenchymal stem cells repair femoral Eur J Immunol 15: 168-173, 1985. segmental defect in rats. J Orthop Res 21: 44-53, 2003. 36. Javazon EH, Colter DC, Schwarz EJ and Prockop DJ: Rat 14. Gysin R, Wergedal JE, Sheng MH, Kasukawa Y, Miyakoshi N, marrow stromal cells are more sensitive to plating density and Chen ST, Peng H, Lau KH, Mohan S and Baylink DJ: Ex vivo expand more rapidly from single-cell-derived colonies than gene therapy with stromal cells transduced with a retroviral human marrow stromal cells. Stem Cells 19: 219-225, 2001. vector containing the BMP4 gene completely heals critical size 37. Camper L, Hellman U and Lundgren-Akerlund E: Isolation, calvarial defect in rats. Gene Ther 9: 991-999, 2002. cloning, and sequence analysis of the integrin subunit alpha10, a 15. Yamazaki M, Nakajima F, Ogasawara A, Moriya H, Majeska RJ, beta1-associated collagen binding integrin expressed on and Einhorn TA: Spatial and temporal distribution of CD44 and chondrocytes. J Biol Chem 273: 20383-20389, 1998. osteopontin in fracture callus. J Bone Joint Surg Br 81: 508-515, 38. Camper L, Holmvall K, Wangnerud C, Aszodi A and 1999. Lundgren-Akerlund E: Distribution of the collagen-binding 16. Asahina I, Sampath TK, Nishimura I and Hauschka PV: Human integrin alpha10beta1 during mouse development. Cell Tissue osteogenic protein-1 induces both chondroblastic and Res 306: 107-116, 2001. osteoblastic differentiation of osteoprogenitor cells derived from 39. Mayer H, Bertram H, Lindenmaier W, Korff T, Weber H and newborn rat calvaria. J Cell Biol 123: 921-933, 1993. Weich H: Vascular endothelial growth factor (VEGF-A) 17. Jackson RA, McDonald MM, Nurcombe V, Little DG and expression in human mesenchymal stem cells: autocrine and Cool SM: The use of heparan sulfate to augment fracture repair paracrine role on osteoblastic and endothelial differentiation. J in a rat fracture model. J Orthop Res 24: 636-644, 2006. Cell Biochem 95: 827-839, 2005. 18. Li J, Ahmad T, Bergstrom J, Samnegard E, Erlandsson-Harris H, 40. Hoffmann A, Czichos S, Kaps C, Bachner D, Mayer H, Ahmed M and Kreicbergs A: Differential bone turnover in an Kurkalli BG, Zilberman Y, Turgeman G, Pelled G, Gross G and angulated fracture model in the rat. Calcif Tissue Int 75: 50-59, Gazit D: The T-box transcription factor Brachyury mediates 2004. cartilage development in mesenchymal stem cell line C3H10T1/2. 19. Yanada S, Ochi M, Adachi N, Nobuto H, Agung M and J Cell Sci 115: 769-781, 2002. Kawamata S: Effects of CD44 antibody- or RGDS peptide-im- 41. Xu J, Wang W, Ludeman M, Cheng K, Hayami T, Lotz JC and mobilized magnetic beads on cell proliferation and chondrogenesis of Kapila S: Chondrogenic differentiation of human mesenchymal mesenchymal stem cells. J Biomed Mater Res A 77: 773-784, 2006. stem cells in three-dimensional alginate gels. Tissue Eng 14: 20. Bruder SP, Jaiswal N, Ricalton NS, Mosca JD, Kraus KH and 667-680, 2008. Kadiyala S: Mesenchymal stem cells in osteobiology and applied 42. Zhao Q, Eberspaecher H, Lefebvre V and de Crombrugghe B: bone regeneration. Clin Orthop Relat Res 355: S247-S256, 1998. Parallel expression of Sox9 and Col2a1 in cells undergoing 21. Ju YJ, Muneta T, Yoshimura H, Koga H and Sekiya I: Synovial chondrogenesis. Dev Dyn 209: 377-386, 1997. mesenchymal stem cells accelerate early remodeling of tendon- 43. Okubo Y and Reddi AH: Thyroxine downregulates Sox9 and bone healing. Cell Tissue Res 332: 469-478, 2008. promotes chondrocyte hypertrophy. Biochem Biophys Res 22. Hadjiargyrou M, Lombardo F, Zhao S, Ahrens W, Joo J, Ahn H, Commun 306: 186-190, 2003. Jurman M, White DW and Rubin CT: Transcriptional profiling 44.Akiyama H, Chaboissier MC, Martin JF, Schedl A and of bone regeneration. Insight into the molecular complexity of de Crombrugghe B: The transcription factor Sox9 has essential wound repair. J Biol Chem 277: 30177-30182, 2002. roles in successive steps of the chondrocyte differentiation 23. Ahmed N, Vogel B, Rohde E, Strunk D, Grifka J, Schulz MB pathway and is required for expression of Sox5 and Sox6. and Grassel S: CD45-positive cells of haematopoietic origin Genes Dev 16: 2813-2828, 2002. enhance chondrogenic marker gene expression in rat marrow 45. De Crombrugghe B, Lefebvre V, Behringer RR, Bi W, stromal cells. Int J Mol Med 18: 233-240, 2006. Murakami S and Huang W: Transcriptional mechanisms of 24. Johnstone B, Hering TM, Caplan AI, Goldberg VM and Yoo JU: chondrocyte differentiation. Matrix Biol 19: 389-394, 2000. In vitro chondrogenesis of bone marrow-derived mesenchymal 46. Schmid TM, Popp RG and Linsenmayer TF: Hypertrophic progenitor cells. Exp Cell Res 238: 265-272, 1998. cartilage matrix. Type X collagen, supramolecular assembly, 25. Hauselmann HJ, Fernandes RJ, Mok SS, Schmid TM, Block JA, and calcification. Ann NY Acad Sci 580: 64-73, 1990. Aydelotte MB, Kuettner KE and Thonar EJ: Phenotypic 47. Zheng Q, Zhou G, Morello R, Chen Y, Garcia-Rojas X and Lee B: stability of bovine articular chondrocytes after long-term culture Type X collagen gene regulation by Runx2 contributes directly in alginate beads. J Cell Sci 107: 17-27, 1994. to its hypertrophic chondrocyte-specific expression in vivo. J 26. Ginzinger DG: Gene quantification using real-time quantitative Cell Biol 162: 833-842, 2003. PCR: an emerging technology hits the mainstream. Exp Hematol 48. Mwale F, Stachura D, Roughley P and Antoniou J: Limitations of 30: 503-512, 2002. using aggrecan and type X collagen as markers of chondrogenesis 27. Pfaffl MW: A new mathematical model for relative quantification in mesenchymal stem cell differentiation. J Orthop Res 24: in real-time RT-PCR. Nucleic Acids Res 29: e45, 2001. 1791-1798, 2006. 745-755 23/4/2009 01:08 ÌÌ Page 755

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 23: 745-755, 2009 755

49. Pelttari K, Steck E and Richter W: The use of mesenchymal 59. Iba O, Matsubara H, Nozawa Y, Fujiyama S, Amano K, Mori Y, stem cells for chondrogenesis. Injury 39: S58-S65, 2008. Kojima H and Iwasaka T: Angiogenesis by implantation of 50. Silva WA Jr, Covas DT, Panepucci RA, Proto-Siqueira R, peripheral blood mononuclear cells and platelets into ischemic Siufi JL, Zanette DL, Santos AR and Zago MA: The profile of limbs. Circulation 106: 2019-2025, 2002. gene expression of human marrow mesenchymal stem cells. 60. Nakagawa H, Komorita N, Shibata F, Ikesue A, Konishi K, Stem Cells 21: 661-669, 2003. Fujioka M and Kato H: Identification of cytokine-induced 51. Mannello F, Tonti GA, Bagnara GP and Papa S: Role and neutrophil chemoattractants (CINC), rat GRO/CINC-2 alpha function of matrix metalloproteinases in the differentiation and and CINC-2 beta, produced by granulation tissue in culture: biological characterization of mesenchymal stem cells. Stem purification, complete amino acid sequences and characterization. Cells 86: 475-481, 2005. Biochem J 301: 545-550, 1994. 52. Cronwright G, Le Blanc K, Gotherstrom C, Darcy P, Ehnman M 61. Quirici N, Soligo D, Bossolasco P, Servida F, Lumini C and and Brodin B: Cancer/testis antigen expression in human Deliliers GL: Isolation of bone marrow mesenchymal stem cells mesenchymal stem cells: down-regulation of SSX impairs cell by anti-nerve growth factor receptor antibodies. Exp Hematol migration and matrix metalloproteinase 2 expression. Cancer 30: 783-791, 2002. Res 65: 2207-2215, 2005. 62. Yaghoobi MM and Mowla SJ: Differential gene expression 53. Tuckermann JP, Pittois K, Partridge NC, Merregaert J and pattern of neurotrophins and their receptors during neuronal Angel P: Collagenase-3 (MMP-13) and integral membrane differentiation of rat bone marrow stromal cells. Neurosci Lett protein 2a (Itm2a) are marker genes of chondrogenic/ 397: 149-154, 2006. osteoblastic cells in bone formation: sequential temporal, and 63. Yada M, Yamaguchi K and Tsuji T: NGF stimulates differentiation spatial expression of Itm2a, alkaline phosphatase, MMP-13, and of osteoblastic MC3T3-E1 cells. Biochem Biophys Res Commun osteocalcin in the mouse. J Bone Miner Res 15: 1257-1265, 205: 1187-1193, 1994. 2000. 64. Mezquita P, Parghi SS, Brandvold KA and Ruddell A: Myc 54. Stetler-Stevenson WG and Seo DW: TIMP-2: an endogenous regulates VEGF production in B cells by stimulating initiation inhibitor of angiogenesis. Trends Mol Med 11: 97-103, 2005. of VEGF mRNA translation. Oncogene 24: 889-901, 2005. 55. Kinnaird T, Stabile E, Burnett MS, Shou M, Lee CW, Barr S, 65. Sherer TB, Neff PS and Tuttle JB: Increased nerve growth Fuchs S and Epstein SE: Local delivery of marrow-derived factor mRNA stability may underlie elevated nerve growth stromal cells augments collateral perfusion through paracrine factor secretion from hypertensive vascular smooth muscle mechanisms. Circulation 109: 1543-1549, 2004. cells. Brain Res Mol Brain Res 62: 167-174, 1998. 56. Liu CH and Hwang SM: Cytokine interactions in mesenchymal 66. Sherer TB, Neff PS, Hankins GR and Tuttle JB: Mechanisms of stem cells from cord blood. Cytokine 32: 270-279, 2005. increased NGF production in vascular smooth muscle of the 57. Pufe T, Harde V, Petersen W, Goldring MB, Tillmann B and spontaneously hypertensive rat. Exp Cell Res 241: 186-193, Mentlein R: Vascular endothelial growth factor (VEGF) induces 1998. matrix metalloproteinase expression in immortalized chondrocytes. J Pathol 202: 367-374, 2004. 58. Rehman J, Li J, Orschell CM and March KL: Peripheral blood ‘endothelial progenitor cells’ are derived from monocyte/ macrophages and secrete angiogenic growth factors. Circulation 107: 1164-1169, 2003.